Abstract

Antisense oligodeoxynucleotides (ODN) have been used to inhibit the function of a number of structurally defined neurotransmitter receptorsin vivo by transiently disrupting their expression in the CNS. However, issues concerning the cellular and molecular mechanisms of these ODN often raise questions about the specificity of such ODN-mediated “knock-down” of target proteins. This study sought to extend our in vivo “knock-down” of the deltaopioid receptor (DOR) by targeting this receptor in the NG 108-15 cells with an antisense ODN for the DOR and by using a polyclonal antibody raised against this receptor to determine the efficiency and selectivity of the antisense ODN in inhibiting expression of the DOR. By fluorescence tagging the ODN and immunofluorescence labeling the DOR, we monitored the uptake efficiency of the ODN and the DOR density in individual cells that had been treated with the antisense ODN or with a mismatch control. Quantitative fluorescence image analysis showed that the uptake of ODN by NG 108-15 cells was time- and concentration-dependent and that it was not uniform within a population. Treatment with the antisense ODN elicited an inverse correlation between DOR immunoreactivity and the ODN fluorescence in individual cells. No correlation was found in cells treated with the mismatch control. These findings suggest that the antisense ODN-mediated “knock-down” of the DOR is governed by the sequence specificity of the ODN and the efficiency of its uptake by the target cells in a time- and concentration-dependent manner. These data provide further evidence in support of the selectivity of antisense ODN targeting and the utility of these molecules as an effective tool in neuropharmacological studies.

Endogenous opioid peptides exert their biological activity via the activation of at least three types of opioid receptors, which have been classified pharmacologically as delta, mu andkappa (Wood, 1982). In addition, pharmacological evidence primarily from in vivo studies of selective opioid ligands has revealed the existence of subtypes for these receptors, further confounding the molecular complexity of opioid action (Mattia et al., 1991; Pick et al., 1991; Lai et al., 1994a). The identification of three discrete genes that encode the three opioid receptors not only confirms the molecular basis of the heterogeneity of the opioid receptors but also provides the means to extend the pharmacological analysis of opioid action at the molecular level (Evans et al., 1992; Kieffer et al., 1992;Thompson et al., 1993; Meng et al., 1993). The deduced primary structure of the three opioid receptors, and their putative secondary structures, as predicted, are closely related to the superfamily of guanine nucleotide regulatory protein (G protein)-coupled receptors. Furthermore, the three receptor polypeptides are highly homologous, with hydrophobic regions that are structurally well conserved. On the other hand, discrete regions, including the amino and carboxyl terminal domains, contain sequences that are unique to each receptor. These discrete regions thus serve as “markers” that distinguish the cloned receptor types, and they have been particularly useful for anatomical localization analysis (Mansouret al., 1994), the design of deletion and chimeric mutation studies to examine ligand interaction with the receptors (Surrattet al., 1994; Xue et al., 1994; Fukuda et al., 1995) and the targeting of specific receptors by antisense strategy (Lai et al., 1994b; Standifer et al., 1994; Bilsky et al., 1996).

We previously explored the use of antisense ODN to transiently and reversibly inhibit the expression of the cloned DOR in mouse brain (Laiet al., 1994b; Bilsky et al., 1996). Antisense ODN are short, synthetic, single-stranded DNA whose mode of action is through hybridization to complementary sequences in the target gene or its messenger RNA; the latter results in the formation of an RNA/DNA duplex that disrupts the normal translation of that gene and/or leads to degradation of the messenger RNA by RNaseH (for review see Crooke, 1992 and Wahlestedt, 1994). Consequently, these molecular events result in a reduction in the level, or “knock-down,” of the protein product. By virtue of the high affinity and specificity of the ODN for their target sequences, the resultant “knock-down” of the target protein could be orders of magnitude greater in specificity than conventional antagonists and is directly correlated with the known structural characteristics of that protein. In our studies, an antisense ODN that is specific to the cloned DOR selectively inhibited the supraspinal antinociceptive effect of [D-Ala2,Glu4] deltorphin, adelta-2 selective agonist, but had no effect on the antinociceptive action of the delta-1 receptor agonist, [D-Pen2,D-Pen5]enkephalin (Lai et al., 1994b; Bilsky et al., 1996). The ability of this antisense ODN to differentiate the antinociceptive effects of the two pharmacologically defined, subtype-selective agonists implicates a structural distinction between these putative receptor subtypes. Treatment with a mismatch control ODN, on the other hand, had no effect on the antinociceptive response to these drugs, which suggests that the effect of the antisense ODN was specific to its sequence rather than due to some nonspecific effect of the ODN. Antisense ODN against the DOR also had no effect on mu or kappa selective agonists (Bilsky et al., 1996). Furthermore, treatment of mice with the ODN did not produce behavioral toxicity, changes in feeding or alterations in base-line nociceptive threshold.

Thus the in vivo effect of the antisense ODN to the cloned DOR is consistent with previous evidence for the existence ofdelta opioid receptor subtypes. On the other hand, a number of issues related to the mechanism of antisense targeting remain unresolved. In particular, it is presumed that the effectiveness of the antisense ODN-mediated “knock-down” depends on its stability, its translocation into the cells that express the target protein and the efficiency with which the antisense ODN suppresses the synthesis of that protein. These issues have not been addressed in our studies using antisense ODN to target the delta opioid receptors in the brain and spinal cord of mice. This communication describes a series of experiments in which we extend our analysis by targeting the DOR in the NG 108-15 cells with the DOR-specific antisense ODN and use a polyclonal antibody that we raised against the DOR to determine the efficiency and selectivity of the antisense ODN-mediated “knock-down” of the DOR. Our findings suggest that antisense ODN-mediated “knock-down” of the target receptor is selective for the target protein. The reduction in the level of the target receptor is correlated with the sequence specificity and the efficiency of its uptake by the target cells in a time- and concentration-dependent manner. These data provide further evidence of the utility of antisense targeting as an effective tool in neuropharmacological studies.

Materials and Methods

Materials.

The cDNA for the mouse DOR was a gift from Dr. Chris Evans (University of California, Los Angeles). The cDNA for the rat KOR and the rat MOR were a gift from Dr. Huda Akil (University of Michigan); [3H]diprenorphine (39 Ci/mmol) and [3H]naltrindole (34.7 Ci/mmol) were from DuPont NEN (Wilmington, DE); tissue culture reagents were from Gibco BRL (Gaithersburg, MD).

Production of polyclonal antibodies to a fusion protein containing the C-terminal peptide of DOR.

A NotI/BalI fragment (nucleotides 1040–1225), which contains the last 105 bp of the coding region of the DOR, was subcloned into the fusion protein expression vector, pGEX-4T-3 (Pharmacia, Piscataway, NJ) to form an in-frame, contiguous coding sequence with that for GST. Transformation ofE. coli with this recombinant DNA resulted in clonal transformants that expressed a high level of a 30-kDa polypeptide, which is made up of a 26-kDa moiety of GST and a 4-kDa moiety of the C-terminal peptide of the DOR. This fusion protein was affinity purified, and 200 to 500 μg/ml was mixed with an equal volume of RIBI adjuvant and injected s.c. into a New Zealand White rabbit. The first boost was given 14 days later, followed by subsequent boosts every 4 weeks thereafter.

Stable expression of the DOR, MOR and KOR.

The cDNA for the three receptors were subcloned into eukaryotic expression vectors (DOR in LK-444; MOR and KOR in pCMV) and transfected into the mouse hippocampal neuroblastoma cell line HN9.10 (Lee et al., 1992) by the calcium phosphate precipitation method. Stable clonal cell lines were selected by neomycin resistance (Geneticin, 1 mg/ml) and maintained in selection medium for at least 10 passages (2 months). Receptor density was monitored regularly by radioligand binding analysis.

Immunocytochemical analysis.

Cells were maintained in 75-cm2 flasks in a humidified atmosphere with 95% air and 5% CO2. NG 108-15 cells were cultured in 5% fetal calf serum/5% newborn calf serum/45% Hams F-12/45% DMEM/100 U ml−1 penicillin/100 μg ml−1 streptomycin. Transfected and nontransfected HN9.10 cells were maintained in 5% fetal calf serum/5% newborn calf serum/90% DMEM/1 mMl-glutamine/100 U ml−1 penicillin/100 μg ml−1 streptomycin. For experiments, cells were seeded onto sterile cover slips contained in 60 mm petri dishes at 50,000 to 100,000 cells/plate 24 hr before the experiments. For experiments in which cells were incubated with ODN, the cells were initially seeded onto cover slips at 100,000 cells/plate in normal media 24 hr before treatment. On the day of the experiments, the cells were washed twice with serum-free medium, and the culture media were replaced with serum-free medium containing ODN at concentrations as specified. The cells were maintained in serum-free conditions throughout the treatment with ODN.

For immunostaining, the medium was aspirated and the cells washed twice with serum-free medium. Immunolabeling was carried out as described previously (Lynch et al., 1991). Briefly, the cells on the cover slips were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. The cells were then incubated with a 1:10 dilution of the antiserum in the presence of 0.1 mg/ml GST for 72 hr at 4°C. The cells were then washed three times with 2 × SSC/0.05% Triton X-100 and incubated with a FITC-conjugated goat anti-rabbit IgG (Vector, Burlingame, CA) for 45 min at room temperature. The cover slips were washed and mounted onto slides with 0.1% p-phenylene diamine dissolved in 50% glycerol.

Oligodeoxynucleotide treatment of NG 108-15 cells.

Texas-red conjugated ODN. The ODN has the following sequence: 5′-CTG TGG CCC CTT GCC GCT GC-3′, which is complementary to the mismatch control sequence for the cloned DOR from NG 108-15 cells (see below). The ODN was synthesized by solid-phase procedure, conjugated with Texas-red at the 5′ end of the ODN and purified by reverse-phase HPLC (Midland Certified Reagent Co., Midland, TX). This Texas-red ODN was reconstituted in nuclease-free water and stored in the dark at 4°C. Cover slips on which NG 108-15 cells were grown were inverted on 40 μl of medium. The medium consisted of Texas-red ODN in serum-free medium at a final concentration of 0.5 μM or 5 μM. The cells on inverted cover slips were incubated in a humidified chamber for 8, 16 and 24 hr, after which time the cells were washed with phosphate buffered saline, fixed with 4% paraformaldehyde and mounted with p-phenylene diamine.

Antisense and mismatch ODN. Antisense (5′-GCA CGG GCA GAG GGC ACC AG-3′; complementary to nucleotide 7-26 of the DOR coding region) and mismatch (5′-GCA GCG GCA AGG GGC CAC AG-3′) ODN to the DOR were used for these experiments. These two sequences, as well as that of the Texas-red-tagged ODN, were screened through the Genbank Database to ensure that these sequences were not likely to cross-react with other known gene sequences. Cells were treated with the antisense or the mismatch ODN at a final concentration of 5 μM for a total of 4 days, during which time the ODN-containing medium was refreshed daily. The dosage was based on the uptake of the Texas-red ODN, and the duration of treatment was based on our previous in vivoantisense treatment, which showed both a maximal functional inhibition and a reduction in the number of supraspinal delta opioid receptors in the antisense ODN-treated, but not the mismatch ODN-treated mice. On day 5, the cells were rinsed briefly and incubated for 24 hr with 5 μM of the Texas-red-ODN as described above. At the end of this incubation, the cells were processed for immunocytochemical analysis as described above.

Uptake of eosin-labeled dextran by the NG 108-15 cells.

Cells were incubated overnight with 50 μg/ml of eosin-dextran (MW 70,000, fixable; Molecular Probes, Eugene, OR) after incubation with the Texas-red ODN. Ability to incorporate the dextran into vesicles by pinocytosis was taken as indicative of cell viability. These cells were rinsed and then incubated with serum-free medium containing dextran.

Fluorescence microscopy.

Fluorescence images were acquired using a Photometrics liquid-cooled CCD camera attached to an Olympus IMT-2 inverted microscope equipped with an Olympus 60X 1.4 NA objective and 6.7X eyepiece. This camera has a linear response up to 5 × 105 counts/image element. An electronic shutter under computer control was utilized to regulate exposure time. Standard optics (Omega Optical, Brattleboro, VT) for FITC included a 10-nm band pass excitation filter centered at 480 nm and a 20-nm band pass emission filter centered at 520 nm. For Texas-red, the excitation and emission were centered at 585 nm and 610 nm, respectively. For eosin, excitation was centered at 525 nm and emission at 545 nm. The digitized output of the camera was stored on a 386 based microcomputer. Image analysis was performed using customized software on a Silicon Graphics IRIS 10/900. The fluorescence intensity within a single cell was quantified by acquiring images of all cells with a constant exposure time. This exposure time (for our studies, it was set at 2 sec) was chosen to provide images with fluorescence intensities well above background [>500 integrated optical density (IOD)] without causing saturation of imaging elements. Thus all measurements were well within the linear response range of the CCD camera.

Cells were selected for analysis on the basis of their Texas-red fluorescence such that they represented the full range of Texas-red intensities observed within that population. A second image was then acquired of the FITC fluorescence in the same cells. The intensity of fluorescence was determined for each cell by tracing around the cell border of each cell and analyzing total IOD within the delineated area. Fluorescence intensity of a cell was then corrected for noncell background fluorescence. This was determined by using a standard 30 × 30 pixel square to sample fluorescence from at least four different out-of-cell areas of the image; the average fluorescence intensity then defined the noncell background. Specific labeling was defined as a level of in-cell fluorescence above the noncell background. NG 108-15 cells do not exhibit significant autofluorescence at the Texas-red wavelength; however, the cells do express significant autofluorescence over the fluorescence window. Specific labeling was expressed as AUF per unit area (μm2). For immunofluorescence of DOR, nonspecific labeling was defined as the in-cell fluorescence (above the noncell background) of cells stained with the secondary fluorophore-labeled antibody alone.

Estimated molecular density of Texas-red ODN.

Because ODN and fluorophore have a stoichiometric ratio of 1:1, an estimate of molecular density was calculated from the measured fluorescence intensity through a sample calibration procedure as described previously (Lynch et al., 1996). Briefly, droplets of fluorophore approximately 0.2 μm in diameter were injected into a layer of mineral oil on the microscope stage. Images of the droplets were acquired, and the exact diameter of the droplet was measured. Because the concentration of a specific molecule in the solution was known, and the volume determined, a value for IOD/molecule could be calculated from the measured fluorescence. For Texas-red ODN, the conversion factor was 4 × 10−2 IOD/molecule, which was used to estimate the molecular density of the ODN (in atmol/μm2).

Confocal microscopy.

Confocal images for presentation were also acquired using a Leica TCS-4D laser scanning confocal microscope and SCANWARE software.

Radioligand binding analysis.

Cell membranes from nontransfected or transfected HN9.10 cells, or with NG 108-15 cells, were resuspended in 50 mM Tris/5 mM MgCl2, pH 7.2. Saturation binding analyses were carried out in the Tris/MgCl2 buffer supplemented with 0.1 mM PMSF and 1 mg/ml bovine serum albumin in a final volume of 1 ml and 27 μg of membrane protein/assay tube. Nonspecific binding of the radioligand was defined as that in the presence of 10 μM naloxone. Membranes were incubated with [3H]naltrindole for 5 hr or with [3H]diprenorphine for 3 hr at room temperature. The reaction was terminated by rapid filtration through Whatman GF/B filters, followed by five washes with ice-cold saline. The radioactivity was determined by liquid scintillation counting.

Results

Polyclonal antibody against the cloned DOR from mouse.

Immune serum produced from the fusion protein displayed strong immunoreactivity with the 30-kDa fusion protein in the presence of 0.1 mg/ml GST by Western analysis (data not shown). Immunocytochemical staining of the NG 108-15 cells with the antiserum showed a granular staining throughout the cytoplasm, whereas the nucleus exhibited little staining (fig. 1). Furthermore, immunocytochemical analysis of three transfected cell lines that expressed the cloned DOR (Evans et al., 1992), MOR (Thompson et al., 1993) and KOR (Meng et al., 1993) showed that the antibody did not cross-react with the MOR or KOR. These cell lines constitutively express the opioid receptors at a density of 5.9 pmol/mg protein (n = 2) for DOR (DORLKHN-8) based on [3H]naltrindole binding, 1.7 pmol/mg protein (n = 2) for MOR (MORCHN-1) and 2.0 pmol/mg protein (n = 3) for KOR (KORCHN-8) based on [3H]diprenorphine binding. Nontransfected HN9.10 cells exhibited negligible levels of [3H]diprenorphine binding. Incubation of DORLKHN-8 cells with the antibody resulted in a granular cytoplasmic staining of these cells similar to that seen in the NG 108-15 cells. Concurrent staining of nontransfected HN9.10 cells, MORCHN-1 and KORCHN-8 cells with the same antibody preparation showed a small degree of staining above background. The result of the analysis of the intensity of fluorescence in these cell lines is summarized in figure 2.

Immunofluorescence image of NG 108-15 cells. The cells were incubated with 1:10 dilution of the anti-DOR antiserum and an FITC conjugated secondary antibody as described in “Materials and Methods.” Magnification: 400×. Scale bar corresponds to 10 μm.

Oligodeoxynucleotide (ODN) treatment of NG 108-15 cells.

Our initial experiments showed that NG 108-15 cells remained viable in the absence of serum over a period of several days if they were initially established at relatively high density (50% confluency) in serum containing medium and were subsequently maintained in serum-free medium that was refreshed daily. These culturing conditions were subsequently used for all our experiments in which the cells were treated with ODN to circumvent the lability of ODN in serum (Akhtar et al., 1991). Cells cultured under these conditions did not actively divide; overnight loading of the cells with eosin-dextran showed that the cells were metabolically viable and expressed substantial amounts of DOR immunoreactivity (see below).

Texas-red ODN uptake by the cells. When the NG 108-15 cells were incubated with the Texas-red ODN to monitor the uptake of ODN by these cells, we observed that the cells accumulated the ODN in a time- and concentration-dependent manner (fig. 3). On the basis of the measured fluorescence intensities shown in figure 3, and a conversion factor of 4 × 10−2 IOD/molecule of the Texas-red ODN, we estimated the molecular density of the tagged ODN that accumulated in these cells over time. Over a 24-hr incubation with 0.5 μM of tagged ODN, the estimated molecular density (EMD; mean ± S.E.M. in atmol/μm2) of ODN was 0.06 ± 0.003, 0.08 ± 0.005 and 0.41 ± 0.24 after 8, 16 and 24 hr of incubation, respectively (fig. 3A). This time-dependent increase in the EMD, however, did not reach statistical significance (P > .2; repeated-measures ANOVA). Incubation with 5 μM of tagged ODN yielded a significant increase in the EMD (mean ± S.E.M. in atmol/μm2) of ODN: 0.07 ± 0.02, 0.48 ± 0.24 and 2.33 ± 0.35 after 8, 16 and 24 hr, respectively (P < .0001; repeated-measures ANOVA) (fig. 3B). Furthermore, cells within a population exhibited a highly variable level of accumulation of the tagged ODN after 24 hr of incubation with 5 μM ODN (figs. 3B; 4). In three separate cultures, the EMD (in atmol/μm2) ranged from 0.18 to 7.01 (n = 16), from 0.24 to 3.36 (n = 13) and from 0.17 to 2.97 (n = 27), which represents up to a 40-fold difference in the EMD of ODN. The mean value of EMD was 1.33 ± 0.16 atmol/μm2 (n = 57), which corresponded to a mean fluorescence intensity of 32,000 ± 3750 AUF/μm2.

Time course and concentration dependence of uptake of Texas-red ODN by the NG 108-15 cells in serum-free conditions. Each point represents the AUF (in thousands)/μm2 of a single cell. A) 0.5 μM of ODN. Total number of cells sampled per time-point is 11. B) 5 μM of ODN. Total number of cells sampled per time-point is 21.

Confocal fluorescence image of NG 108-15 cells that had been incubated with 5 μM of Texas-red ODN for 24 hr in serum-free medium. Magnification: 100×.

Irrespective of the levels of Texas-red staining, and thus of the level of ODN accumulated in these cells, all the cells that were examined also accumulated dextran (fig. 5). The ODN was found initially in endosome-like structures, and over time it was localized to both the cytoplasm and the nucleus. The uptake characteristics of the tagged ODN, as well as dextran accumulation, were qualitatively similar in cells that had been preincubated with mismatch ODN (nontagged) for up to 4 days, which indicates that the cells were not adversely affected by prolonged exposure to these ODN (data not shown).

Antisense and mismatch ODN pretreatment of the cells.Experiments were carried out to evaluate the relationship between the level of antisense ODN uptake and expression of DOR. Because longer periods of ODN treatment are required to block the expression (3–4 days) than to monitor uptake, cells were first incubated with 5 μM ODN daily for 4 days and then labeled for uptake by incubation with 5 μM of Texas-red ODN for 24 hr. Under this treatment paradigm, we found that cells that accumulated substantial amounts of Texas-red ODN over 24 hr after DOR antisense ODN treatment consistently exhibited a lower level of DOR antibody staining (fig. 6). Because we could not determine directly the stoichiometric ratio of FITC-conjugated second antibody to DOR molecules, we were not able to correlate directly the density of DOR with tagged ODN uptake. Thus we made a semiquantitative comparison between ODN accumulation and DOR density by correlating the level of Texas-red and FITC fluorescence in the same cell (fig. 7). We found that over the range of 4560 to 31,700 AUF/μm2 of Texas-red fluorescence, there was a significant inverse correlation between the immunoreactivity of DOR and the amount of Texas-red ODN accumulated in these cells (P < .01, linear regression, correlation coefficient = −0.71) (fig.7A). Furthermore, cells that accumulated over 25,000 AUF/μm2 of Texas-red all showed markedly reduced DOR immunoreactivity averaging 2080 ± 159 AUF/μm2(n = 11). In contrast, no correlation was observed in cells that had been pretreated with the mismatch control ODN before labeling with Texas-red ODN (P = .06; two-tailed ttest) (figs. 7B, 8). Among the cells that exhibited over 25,000 AUF/μm2 of Texas-red, the average DOR staining was 17,300 ± 2870 AUF/μm2 (n = 6). As mentioned above, the mean fluorescence intensity of Texas-red ODN in cells loaded with 5 μM of the ODN was 32,000 ± 3750 AUF/μm2. Thus it appears that the ability of a cell to accumulate a large amount of ODN (within the upper 50% of the observed range of ODN densities) correlates with up to 88% reduction in DOR expression after antisense ODN treatment when compared with mismatch ODN-treated cells. Qualitatively, the prevalence of cells that accumulated within the upper range of Texas-red ODN was 50% to 80% of a typical culture.

Discussion

This study examines the selectivity of antisense ODN in mediating a “knock-down” of its target protein. By employing fluorescence imaging and computer-assisted image analysis, we monitored the level of the antibody/DOR complex and that of a fluorescence-labeled ODN simultaneously in individual cells. These results suggest a direct relationship between the uptake of antisense ODN for the DOR and a reduction of its target, the endogenous DOR in the NG 108-15 cells. The effects of the antisense ODN are sequence-dependent, because a mismatch control ODN did not bring about any change in the level of DOR in these cells.

Fusion proteins are now well established as a means of efficiently producing antigens for the generation of polyclonal, as well as monoclonal, antibodies against defined epitopes from specific proteins. A number of other antibodies also have been developed recently for the opioid receptors and applied in a variety of immunohistochemical analyses (Arvidsson et al., 1995a,b,c). The polyclonal antibodies generated from the fusion protein containing the C-terminal epitope of the DOR display reactivity toward both the fusion protein and NG 108-15 cells (fig. 1). The polyclonal antibody also does not exhibit significant cross-reactivity to the other opioid receptor types. As shown in figure 2, the transfected cells expressing the DOR exhibited an intensity of fluorescence/μm2 over 6-fold that of nontransfected or transfected cells that express the MOR or the KOR. Both the NG 108-15 cells and transfected cells expressing the DOR exhibited significant cytoplasmic immunoreactivity. Interestingly, this cytoplasmic localization of the DOR resembles the distribution of the receptor in spinal cord and brainstem neurons using a DOR-specific antibody (Arvidsson et al., 1995a). The cellular and molecular basis for the subcellular distribution of the DOR currently remains speculative. Our antibody to the DOR enabled us to monitor the level and distribution of the DOR in experiments in order to examine the effect of transient targeting of this receptor by antisense ODN in individual NG 108-15 cells.

Initial experiments using fluorescence-tagged ODN showed that viable NG 108-15 cells accumulated the ODN in a time- and concentration-dependent manner (fig. 3). These observations are consistent with a number of previous studies in which phosphodiester ODN (Loke et al., 1989; Yakubov et al., 1989) or phosphorothioate ODN (Gaoet al., 1993; Beltinger et al., 1995) were shown to be taken up by a number of different cell lines, and this uptake was thought to be mediated by both adsorptive endocytosis and fluid-phase pinocytosis. On the basis of the purity of the tagged ODN preparation (HPLC purified) and its stability in serum-free conditions (thin-layer chromatography of the incubation medium containing 5 μM Texas-red ODN after 24 hr of incubation with cells did not detect any free dye in the medium), our data indicate that the fluorescence detected in the NG 108-15 cells corresponded directly to the level of intact ODN that had been taken up. Independently of the concentration of the ODN or the duration of incubation, however, the uptake of the ODN was not uniform throughout the cell population (fig. 3). This result argues against a diffusion mechanism for uptake, which again is consistent with previous findings (Loke et al., 1989). The heterogeneity of ODN uptake by the cells was not due to their viability, based on dextran uptake. Moreover, cells that showed intense Texas-red ODN uptake were comparable morphologically under phase-contrast microscopy to those that were less fluorescent throughout all time-points and different concentrations. These data suggest that the cultured cells may accumulate the ODN at a different rate and/or to a different degree; because dead cells do not accumulate ODN (Loke et al., 1989), such rate and degree of accumulation of the ODN may depend on cell metabolism. It should be noted that fluorescence does not necessarily indicate the localization of full-length ODN, because degradation of ODN readily occurs intracellularly (Yakubov et al., 1989; Crooke et al., 1995), but it does directly reflect the amount of tagged ODN that has been accumulated by the cells. Furthermore, fluorescence intensity increased both in the cytoplasm and in the nucleus, which suggests that the ODN had access to both cell compartments—a result similar to observations made with other cell types exposed to ODN (Loke et al., 1989; Gaoet al., 1993; Beltinger et al., 1995; Crookeet al., 1995).

Overall, these findings show that uptake of the ODN was prevalent over 24 hr when the NG 108-15 cells were exposed to 5 μM ODN. Repeated dosing of the cells with the antisense ODN at 5 μM for 4 days, a treatment paradigm that was intended to mimic that employed to successfully “knock-down” DOR in mice, showed that the antisense ODN significantly reduced the overall level of DOR in the NG 108-15 cells when compared with cells that had been treated with the mismatch ODN at the same concentration. DOR antisense ODN, but not the mismatch control, resulted in a 40% reduction in [3H]diprenorphine binding sites, which is consistent with that observed previously (Standifer et al., 1994; Zhouet al., 1994). At the single-cell level, a reduction in the DOR, based on its immunoreactivity, was observed only in cells that had subsequently accumulated a substantial amount of the Texas-red ODN. This outcome establishes a strong physical correlation between the accumulation of the antisense and the Texas-red ODNs and a reduction in the level of DOR. It is interesting that there is a statistically significant inverse correlation between the level of DOR and ODN fluorescence in the antisense ODN-treated cells. However, because of the relatively small sample size, and because the intracellular disposition of ODN over the treatment period is complex, the precise concentration relationship between the antisense ODN and DOR remains to be determined. Nevertheless, the reduction of the DOR was not due to uptake of the Texas-red ODN itself, because its sequence was not specific to the DOR. Furthermore, cells that had been pretreated with antisense ODN or mismatch control ODN were subsequently loaded with the same Texas-red ODN. The reduction in the DOR level also could not be due to toxicity of the ODN, because preliminary studies (discussed above) had ruled out possible changes in DOR expression due to cell viability alone, and, moreover, cells that had been pretreated with the mismatch control ODN exhibited, on average, a much higher level of DOR irrespective of Texas-red ODN accumulation (fig. 7B). These observations suggest, therefore, that the cells were more susceptible to the antisense pretreatment when they exhibited efficient ODN uptake. Indeed, the most obvious observation from cells that had been treated with the antisense ODN was the marked reduction in DOR immunoreactivity when compared with the mismatch ODN-treated cells over the upper range of ODN uptake.

This clear-cut correlation between Texas-red ODN uptake and the DOR immunoreactivity is consistent with the assumption that the efficiency of the Texas-red ODN uptake in the last 24 hr of incubation paralleled the extent to which the cells had continually accumulated the antisense ODN during the 4-day treatment. Further, the rate and/or extent of antisense ODN accumulation was sufficient to abolish completely the expression of the DOR in some cells. The reduction in the level of DOR was specifically due to the antisense sequence for the DOR, because pretreatment with the mismatch ODN did not alter the level of DOR, irrespective of the level of ODN uptake by cells within the population. The selectivity of the DOR antisense ODN for the DOR has also been observed in vivo; treatment with this antisense ODN had no effect on the antinociception of either kappa ormu opioid receptor selective agonists (Bilsky et al., 1996). It should be pointed out that Texas-red-labeled antisense ODN, or its mismatch control, was not used for the 4-day treatment paradigm because of concern that the 5′-end labeling with the fluorophore might alter the affinity of the ODN for its RNA target, and also because of the prohibitive cost of the procedure. Overall, these findings provide direct evidence at the single-cell level that the antisense ODN, but not the mismatch ODN, mediated “knock-down” of the DOR in a highly selective manner. This action of the antisense ODN is governed by the sequence of the ODN and depends critically on the translocation of the ODN into the NG 108-15 cells.